Abstract
The facultative intracellular bacterium Legionella pneumophila employs the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). The endoplasmic reticulum (ER)-resident large fusion GTPase Sey1/atlastin promotes remodeling and expansion of LCVs, and the GTPase is also implicated in the formation of ER-derived lipid droplets (LDs). Here we show that LCVs intimately interact with palmitate-induced LDs in Dictyostelium discoideum amoeba. Comparative proteomics of LDs isolated from the D. discoideum parental strain Ax3 or ⊗sey1 revealed 144 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or ⊗sey1, respectively. Using dually fluorescence-labeled amoeba producing the LCV marker P4C-GFP or AmtA-GFP and the LD marker mCherry-perilipin, we discovered that Sey1 and the L. pneumophila Icm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions. In vitro reconstitution of the LCV-LD interactions using purified LCVs and LDs from D. discoideum Ax3 or ⊗sey1 revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired for ⊗sey1-derived LDs, suggesting that Sey1 regulates LD composition. Palmitate promoted the growth of (i) L. pneumophila wild-type in D. discoideum Ax3 but not in ⊗sey1 mutant amoeba and (ii) L. pneumophila wild-type but not ⊗fadL mutant bacteria lacking a homologue of the E. coli fatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellular L. pneumophila metabolizes 13C-palmitate, and its catabolism was reduced in D. discoideum ⊗sey1 and L. pneumophila ⊗fadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Abbreviations
- Atl
- atlastin
- cryoET
- cryo-electron tomography
- Icm/Dot
- intracellular multiplication/defective organelle trafficking
- LCV
- Legionella-containing vacuole
- LD
- lipid droplet
- GFP
- green fluorescent protein
- MCS
- membrane contact site
- OSBP
- oxysterol binding protein
- PI
- phosphoinositide
- p.i.
- post infection
- Plin
- perilipin
- T4SS
- type IV secretion system
- VAMP
- vesicle-associated membrane protein
- Vap
- VAMP-associated protein